Method of suppressing immuneresponse with 1 substituted-3-(2 pyrimidyl)ureas

ABSTRACT

1-Substituted-3-(2-pyrimidyl)ureas as immunosuppressants and as antiviral agents.

United States Patent [191 Paget [4 11 Sept. 18, 1973 Assignee:

METHOD OF SUPPRESSING IMMUNERESPONSE WITH 1 SUBSTlTUTED-3-(2PYRIMIDYL)UREAS Charles J. Paget, Indianapolis, Ind.

Eli Lilly and Company, Indianapolis, Ind.

Filed: June 9, 1971 Appl. No.: 151,540

Related U.S. Application Data Continuation-impart of Ser. No. 867,079,Oct. 16, 1969, abandoned.

Inventor:

U.S. Cl 424/251, 260/256.4, 260/256.5 Int. Cl A611: 27/00 Field ofSearch 424/251 Urbanski et al., Journal of Medicinal Chemistry, Vol. 10,pages 521525, 1967 Primary Examiner-Richard L. Huff Attorney-Event F.Smith and Kathleen R. Schmoyer [5 7] ABSTRACTl-Substituted-3-(Z-pyrimidyl)ureas as pressants and as antiviral agents.

immunosup- 3 Claims, No Drawings METHOD OF SUPPRESSING IMMUNERESPONSEWITH 1 SUBSTITUTED-3-(2 PYRIMIDYL)UREAS CROSS-REFERENCE TO RELATEDAPPLICATION This is a continuation-in-part of my copending applicationSer. No. 867,079, filed Oct. 16, 1969 now abancloned.

BACKGROUND OF THE INVENTION Recently, immunosuppressant agents have comeinto prominence because of their use incident to transplants of organsfrom one human to another, especially heart transplants and kidneytransplants. It is part of the defense mechanism of humans to removeforeign antigens (in this case, the transplanted organ) by the immunereaction. Thus, in all of the organ transplant operations, it has beennecessary to give large doses ofan immunosuppressant prior to theoperation and continuing thereafter in order to prevent the host fromrejecting the donor organ. The immunosuppressant of choice has beenazathiaprene (US. Pat. No. 3,056,785).

SUMMARY OF THE INVENTION The present invention is concerned most broadlywith compounds of the formula:

In the above and succeeding formulae throughout the presentspecification,

each R independently represents hydrogen, chloro,

or loweralkyl of C -C each R independently represents hydrogen orloweralkyl of C -C Y represents S or O; R represents cycloalkyl of C -Cnaphthyl, phenyl,

or substituted phenyl of the formula:

wherein each R independently represents halo, loweralkyl of C -C orloweralkoxy of C -C R represents cyano, trifluoromethyl, or nitro; mrepresents an integer of from O to 2, both inclusive; and n representsan integer of from to 1, both inclusive, the sum of m and n being 1 or2. In the present specification'and claims, the term halo is used torefer to chloro, bromo, fluoro, and iodo, only.

All of the compounds as thus defined are useful as immunosuppressantsand as antiviral agents. Accordingly, the present invention is directedin part to a method for preventing infection of a mammal subject toattack by Coe virus which method comprises the step of administering themammal an effective dose within the range of from 0.01 to 1,000milligrams of an active agent as above defined per kilogram of mammalbody weight.

For immunosuppression, certain compounds within the broad definition arepreferred. Accordingly, the

present invention is directed, in another part, to a method for loweringthe efficiency of defense by a host mammal against a substancerecognized as a foreign substance and reacted to immunologically whichcomprises the step of administering parenterally to the mammal aneffective dose within the range of from 0.5

to 400 milligrams of an active agent per kilogram of mammal body weight,said active agent being a compound of the formula the formula 5 whereinR represents cycloalkyl of CQ-C naphthyl, or

substituted phenyl of the formula wherein R represents loweralkyl of C Cor loweralkoxy of C -C R represents cyano or trifluoromethyl; and R, R,Y, m and n are employed as defined above.

DETAILED DESCRIPTION OF THE INVENTION All of the compounds of thepresent invention are prepared by reacting a 2-aminopyrimidine compoundof the formula with a carbamoyl chloride or thiocarbamoyl chloride ofthe formula Additionally, those compounds of the present invention whichare of the formula can also be prepared by reacting a 2-aminopyrimidine,as above defined, with an isocyanate or isothiocyanate of the formula Incarrying out the former reaction of 2- aminopyrimidine and the carbamoylchloride or thiocarbamoyl chloride, the reactants are combined withanother, conveniently in the presence of an inert sol-' vent as reactionmedium, and necessarily in the presence of a hydrogen chlorideacceptor-such as an organic tertiary amine. The reaction goes forwardreadily at temperatures over a wide range, such as from C. or less toreflux temperatures. It is preferably conducted by mixing the reactantsat room temperatures and heating the reaction mixture to reflux. Thereaction results in the preparation of the desired product in goodyield, with the hydrochloride salt of the acceptor as by-product.Separation, and if desired purification, are carried out in conventionalprocedures.

In the second synthetic procedure, the 2- aminopyrimidine and isocyanateor isothiocyanate are mixed with one another, conveniently in thepresence of an inert solvent as reaction medium. The reaction goesforward readily at temperatures of from room temperatures to reflux, butis conveniently conducted at reflux. Separation, and if desiredpurification, are carried out in conventional procedures.

The following examples illustrate the synthesis of compounds of thepresent invention. EXAMPLE 1: l-NAPHTl-lYL-3-(2- PYRIMIDYL)UREA In 150milliliters of toluene, 2-aminopyrimidine (2.87 grams; 0.03 mole) andl-naphthyl isocyanate (5.6 grams; 0.03 mole) were mixed, and the mixtureheated to reflux and refluxedfor 8 hours with stirring. The reactionmixture was then cooled and filtered to separate the desiredl-naphthyl-3-(2-pyrimidyl)urea product as a solid, m.p., 246-48C.

EXAMPLES 2-38 Other representative products of the present inventionprepared in accordance with the foregoing example and teachings arelisted in the following table, in which mp. is used as an abbreviationof the term melting point, and m.w., as an abbreviation of the termmolecular weight.

4 TABLE 1 Characterizing Property(Trifluoromethyl)phenyl)-2-pyrimidyl)urea m.p., 223-24C.l-(p-Nitrophenyl)-3-(2-pyrimidyl)-urea m.p., 26870C.l-Phenyl-3-(2-pyrimidyl)urea m.p., 22l-22C. l -Phenyl- I -methyl-3-(2-pyrimidyl )-thiourea m.w., 244 .3 l-( S-(Trifluoromethyl)-m-tolyl)-3-(2- pyrimidyl)urea m.w., 296.3 l -Phenyll ,3dimethyl-3-(2-pyrimidyl)-urea m.w., 242.3 l-(3,4-Dichloropheyl)-3-(2-pyrimidyl)-uream.p., 240-4 1C. l-(m-Tolyl)-3-(2-pyrimidyl)urea m.p., l87-88C.

l-Cyclohexyl-3-ethyl-3-(2-pyrimidyl)urea a m.w., 248.31-(p-Cyanophenyl)-3-(2-pyrimidyl)urea m.p., 273-74C.l-(p-Fluorophenyl)-3-(2-pyrimidyl)urea m.p., 244-45C.l-Phenyl-3-(4,6-dichloro-2-pyrimidyl)thiourea m.w., 299.2

l-(o-Fluorophenyl )-3-(2-pyrimidyl )urea 1-( 2,5-Dichlorophenyl)-3-(2-pyrimidyl)urea l-(p-n-Propylphenyl)-3-(2-pyrimidyl)thiourea m.w.,272.4 l-(o-Tolyl)-3-(2-pyrimidyl)urea m.p., 2l4-l5C.l-(p-lodophenyl)-3-( 2-pyrimidyl)urea m.w., 340.1l-(p-Tolyl)-3-(2-pyrimidyl)urea m.p., 20506 C. l-( p-Nitrophenyl )-3-(4,6-dimethyl-2- pyrimidyl)urea m.w., 287.3l-(3,5-Xylyl)-3-(3-pyrimidyl)urea m.w., 242.3

m.w., 358.8 m.p., 24849C.

m.p., 205-07C.

l-Cyclopentyl-3-(2-pyrimidyl)urea m.w., 206.3l-Cyclooctyl-3-(Lpyrimidyhthiourea m.w., 264.4l-(m-Nitrophenyl)-3-(2-pyrimidyl)urea m.p., 2596lC.l-(4-Nitro-o-tolyl)-3-( 2-pyrimidyl)thiourea m.w., 289.3

(2-pyrimidyl)urea m.w., 338.3 l-Phenyl-3-(4-chloro-6-methyl-2-pyrimidyl)urea m.p., l56-5 7"C. l-( 3-Bromo-5-isopropoxyphenyl )-3-( 2pyrimidyl)urea V m.w., 351.2 I-Naphthyl-3-(4-chloro-6-methyl-2-pyrimidyl)urea m.p., 204-05C. l-( p-Nitrophenyl l -isopropyl-3 2-pyrimidyl)thiourea m.w., 303.3

As stated hereinabove, the compounds of the present invention are usefulin suppressing the immune reaction in mammals. Such suppression includesthe suppression of immune response engendered whenever the mammalianbody forms antibodies in response to the presence of the foreignprotein. Thus, immunosuppressant activity can also be characterized asantiallergic. The practical application of immunosuppressant activity isvaried. As noted above, a prominent application of immunosuppressantactivity is in the transplanting of organs; but immunosuppressantactivity can also be advantageously employed in the therapy of thevarious diseases known collectively as autoimmune" diseases. Althoughthe etiology of these diseases is little understood, it is generallybelieved that an immune reaction is involved. Representative suchautoimmune diseases include auto-immune hemolytic anemia, idiopathicthrombocytopenic purpura, lupus erythematosus, lupoid hepatitis, lupusnephritis, glomeru-v jectable solution--conveniently a physiologicalsaline solution containing one or more of the compounds of the presentinvention. Methods and procedures for preparing such injectablesolutions, and techniques for administering them, are well known in theprior art.

The ability of the compounds of the present invention to suppress immunemechanisms in a host animal was measured by their activity asanti-allergic agents according to the following test. Groups of five-gram Swiss mice were each injected intraperitoneally with standardizedsuspensions of an antigen-in this instance sheep blood cells. Thecompounds were also injected by the intraperitoneal route at varioustimes before and/or after the injection of the sheep blood cells for atotal of three injections of the same amount of compound on eachoccasion. Eight days after injection of the antigen, the mice were bledand the sera from each group pooled. Antibody determinations were madeon the serum pools by a hemaglutination pattern procedure andcomparisons made between treated and control animals. The activity ofthe compounds is given in the following table in terms of the minimumdose of drug administered on each of three occasions that was necessaryto suppress the hemaglutination titer by a four-fold factor in thetreated mice as compared with control titers.

R R Y R Dose in mgJkg. X 3 for Four-fold or Greater Suppression H H Om-CEC H, H H O p-NO,C.H 12.5 H H O C 5 50.0 H H O 3,4-C1,C.H, 50.0 H H OmCH CJh 50.0 H H O o-NO,C.H 50.0 H H O C,H 100.0 H H O p-CNC H 100.0 H HO pFC H 200.0 H H O 2,5-Cl,C-H, 100.0 H H O o-CH,C.H 100.0 H H Om-NO,C.H 200.0 H H O m-CIC H 200.0 4-CH,, 6-Cl H O l-C H, 100.0Azathioprene 100.0

As previously stated, the compounds of the present invention are alsouseful in combatting infections of viral origin in mammals, inparticular in combatting infections due to the Coe virus. In accordancewith this embodiment of the present invention, there is administered toa mammal subject to attack by Coe virus an effective dose within therange of from 0.01 to 1,000 milligrams thereof per kilogram of mammalbody weight. As in the instance of theembodiment of the presentinvention in immunosuppression, it is necessary that the compounds beadministered parenterally, and the comments hereinabove regarding thepreparation of suitable formulations are equally applicable v v Thisembodiment of the present invention is illustrated by a test procedureinvolving mice infected with Coxsacki A21 (Coe) virus. This virus wasisolated by Lanette et al., Am. J. Hyg. 68, 2727 (1959), from humanpatients with mild respiratory illness. In mice, the virus producesmuscle degeneration and flaccid paralysis as well as respiratoryinvolvement. Table 3, which follows, gives for the compounds listedtherein, the ED (effective dose for protecting 50 percent of the animalsfrom the efiects of administered virus at levels which produce lethalinfections in I00 percent of the mice) when the drug is givenintraperitoneally in unit dosage form to mice 3 hours before injectionof the virus. The mouse-adapted Coxsaclti A21 virus employed is usuallyin the form of a 1:10 dilution of the supemate of a 10 percent mousegluteal muscle (obtained from infected mice) suspension. The LD of thesuspension was determined in each case by titration in mice. Thecompounds were administered to the mice as suspensions in 0.25 ml. of asterile water (permissibly containing up to 3 percent of a non-ionicsurfactant) at a series of dose levels from which the ED, could bedetermined. The mice employed were SPF white Swiss mice weighing 11-13grams. Ten mice were used for each drug level, and three-five groups of10 mice each were used as controls. In this procedure, the control miceusually die on the fifth to eighth day after infection, and animalsalive through the 10th day are considered survivors if free ofparalysis. The ED given in the tables were calculated by the method ofReed and Muench, Am. J. Hyg. 27, 493 (1938).

In Table 3, a dash indicates that the compound was not active at thehighest dose tested, usually 128 mg./kg. in the anti-viral test, butpresumably would be active at higher dose levels.

TABLE 3 Essentially like results are obtained when evaluating the otherof the compounds identified hereinabove as Examples 1-38 in the sametest.

Many of the starting materials to be employed in accordance with thepresent invention are known compounds, and all of the same are preparedin known procedures. All of the 2-aminopyrimidine starting materials:

each R independently represents hydrogen, chloro,

or loweralkyl of C -C each R independently represents hydrogen orlowerwherein alkyl of C C Y represents S or O;

R represents cycloalkyl of C -C phenyl, nitrophenyl,

(trifluoromethyl )phenyl,

cyanophenyl,

tolyl, o-tolyl, p-fluorophenyl, m-chlorophenyl, 2,5- dichlorophenyl, or3,4-dichlorophenyl. 2. The method of claim 1 wherein the active agent is3. The method of claim 1 wherein the active agent is phenyl)urea.

1-( 2-pyrimidyl)-3-(p-nitro

2. The method of claim 1 wherein the active agent is1-(2-pyrimidyl)-3-(m-(trifluoromethyl)phenyl)urea.
 3. The method ofclaim 1 wherein the active agent is1-(2-pyrimidyl)-3-(p-nitrophenyl)urea.